Glioblastoma is the most common primary brain cancer in adults and remains a deadly disease. Despite intense research and clinical efforts, its survival has not significantly improved for the past several decades. A major reason for this recalcitrant nature is the tumor?s ability to escape toxicity from currently available therapies, including Temozolomide (TMZ), radiation, and immune-modulation. This resistance depends on the activation of defense mechanisms in response to damage, and is mediated epigenetically by a complex, multi- component machinery that governs the transcriptional status of the tumor cells. Our studies have defined oncogenic proteins EZH2, BMI1 and LSD1 as crucial components of this epigenetic response. We have also demonstrated that they are regulated by specific microRNAs (miR-124, miR-128 and miR-137), co-expressed in normal brain, and simultaneously lost in glioblastoma. The re-expression of these three microRNAs in glioblastoma provides a strong inhibition against the three proteins and results in a very relevant sensitization of tumor cells to stress, both in vitro and in vivo. The overall goal of this proposal is two-fold: first, to characterize the mechanism whereby the co-activation of EZH2, BMI1 and LSD1 mediates tumor survival, establishing its necessary role in this response; Second, to develop a microRNA-mediated strategy to abate this epigenetic shield that is vital to the tumor, to support a gene therapy approach synergistic with current standard therapies. AIM1 proposes experiments to dissect the role of the EZH2/BMI1/LSD1 epigenetic complex in tumor responses against Temozolomide, radiation, and immunotherapy. AIM2 provides novel solutions to design and construct artificial genes that multiply the payload of microRNAs, and which thus function as an ideal gene therapy platform for multi-targeting this epigenetic response. AIM3 investigates the applicability of multi-microRNA-based therapy in animal models of glioblastoma, to address the vital problem of tumor resistance in vivo. INNOVATION: The proposed study to use artificial microRNA clusters for the control of the complex epigenetic tumor survival response is an innovative and heretofore unexplored approach. Also, this proposal introduces strategies to exploit the unique features of microRNAs biology, processing and intercellular trafficking in the perspective of an integrated gene therapy approach. LONG-TERM OBJECTIVE: We seek to meaningfully impact the care of glioblastoma patients through the application of principles of microRNA clustering, epigenetic interference, and synergism with other therapies.